Alzheimer's disease (AD) is the most common form of both senile and presenile dementia in the world and is recognized clinically as relentlessly progressive loss of memory and intellectual function and disturbances in speech (Merritt, 1979, A Textbook of Neurology, 6th edition, pp. 484-489, Lea & Febiger, Philadelphia, which is incorporated herein by reference). Alzheimer's disease begins with mildly inappropriate behavior, uncritical statements, irritability, a tendency towards grandiosity, euphoria, and deteriorating performance at work; it progresses through deterioration in operational judgment, loss of insight, depression, and loss of recent memory; and it ends in severe disorientation and confusion, apraxia of gait, generalized rigidity, and incontinence (Gilroy & Meyer, 1979, Medical Neurology, pp. 175-179, MacMillan Publishing Co., which is incorporated herein by reference,). Alzheimer's disease is found in about 10% of the population over the age of 65 and 47% of the population over the age of 85 (Evans et al., 1989, JAMA, 262:2551-2556, which is incorporated herein by reference).
Alzheimer's Disease is characterized by the accumulation of insoluble, 10 nm filaments containing β-amyloid (Aβ) peptides, localized in the extracellular space of the cerebral cortex and vascular walls. These 40 or 42 amino acid long Aβ peptides are derived from the larger β-amyloid precursor protein (βAPP) through the endopeptidase action of β and γ secretases. In addition, the post-translational action of putative aminopeptidases results in a heterogeneous shortening of the 40 or 42 amino acid long Aβ peptides that either terminate at residue 40 or 42 and, therefore, are designated as AβN-40 and AβN-42. In familial forms of AD, the pathological appearance of the Aβ peptides in the brain is driven by the presence of mutations in the βAPP gene or in the genes coding for the proteins presenilin 1 and 2.
Sporadic AD accounts for more than 95% of the known AD cases. Its etiology, however, remains obscure. An accepted view is that sporadic AD results from the interplay between an individual's genetic factors and the environment, leading to the deposition of Aβ, neurodegeneration, and dementia. Despite this emerging perspective, insufficient effort has been made in identifying factors responsible for Aβ accumulation in the brain.
The etiology of Alzheimer's disease is unknown. Evidence for a genetic contribution comes from several important observations such as the familial incidence, pedigree analysis, monozygotic and dizygotic twin studies, and the association of the disease with Down's syndrome (for review see Baraitser, 1990, The Genetics of Neurological Disorders, 2nd edition, pp. 85-88, which is incorporated herein by reference). Nevertheless, this evidence is far from definitive, and it is clear that other factors are involved.
Alzheimer's Disease is a neurodegenerative disease characterized by a progressive decline of cognitive functions, including loss of declarative and procedural memory, decreased learning ability, reduced attention span, and severe impairment in thinking ability, judgment, and decision making. Mood disorders and depression are also often observed in AD patients. It is estimated that AD affects about 4 million people in the USA and 20 million people worldwide. Because AD is an age-related disorder (with an average onset at 65 years), the incidence of the disease in industrialized countries is expected to rise dramatically as the population of these countries ages.
AD is characterized by the following neuropathological features:                massive loss of neurons and synapses in the brain regions involved in higher cognitive functions (association cortex, hippocampus, amygdala). Cholinergic neurons are particularly affected.        neuritic (senile) plaques that are composed of a core of amyloid material surrounded by a halo of dystrophic neurites, reactive type I astrocytes, and numerous microglial cells (Selkoe, D. J., Annu Rev Neurosci 17:489-517, 1994; Selkoe, D. J., J Neuropathol Exp Neurol 53:438-447, 1994; Dickson, D. W., J Neuropathol Exp Neurol 56:321-339, 1997; Hardy, J. et al., Science 282:1075-1079, 1998; Selkoe, D. J., Cold Spring Harb Symp Quant Biol 61:587-596, 1996, all of which are incorporated herein by reference. The major component of the core is a peptide of 39 to 42 amino acids called the amyloid β protein, or Aβ. Although the Aβ protein is produced by the intracellular processing of its precursor, APP, the amyloid deposits forming the core of the plaques are extracellular. Studies have shown that the longer form of Aβ (Aβ42) is much more amyloidogenic than the shorter forms (Aβ40 or Aβ39).        neurofibrillary tangles that are composed of paired-helical filaments (PHF) (Ray et al., Mol Med Today 4:151-157, 1998; Brion, Acta Neurol Belg 98:165-174, 1998, both of which are incorporated herein by reference). Biochemical analyses revealed that the main component of PHF is a hyper-phosphorylated form of the microtubule-associated protein τ. These tangles are intracellular structures, found in the cell body of dying neurons, as well as some dystrophic neurites in the halo surrounding neuritic plaques.        
Both plaques and tangles are found in the same brain regions affected by neuronal and synaptic loss.
Although the neuronal and synaptic loss is universally recognized as the primary cause of the decline of cognitive functions, the cellular, biochemical, and molecular events responsible for this neuronal and synaptic loss are subject to fierce controversy. The number of tangles shows a better correlation than the amyloid load with the cognitive decline (Albert, Proc Natl Acad Sci USA 93:13547-13551, 1996, which is incorporated herein by reference). On the other hand, a number of studies showed that amyloid can be directly toxic to neurons, resulting in behavioral impairment (Ma et al., Neurobiol Aging 17:773-780, 1996, which is incorporated herein by reference). It has also been shown that the toxicity of some compounds (amyloid or tangles) could be aggravated by activation of the complement cascade, suggesting the possible involvement of inflammatory process in the neuronal death.
Genetic and molecular studies of some familial forms of AD (FAD) have recently provided evidence that boosted the amyloid hypothesis (Ii, Drugs Aging 7:97-109, 1995; Price et al., Curr Opin Neurol 8:268-274, 1995; Hardy, Trends Neurosci 20:154-159, 1997; Selkoe, J Biol Chem 271:18295-18298, 1996, all of which are incorporated herein by reference). The assumption is that since the deposition of Aβ in the core of senile plaques is observed in all Alzheimer cases, if Aβ is the primary cause of AD, then mutations that are linked to FAD should induce changes that, in one way or another, foster Aβ deposition. There are 3 FAD genes known so far (Hardy et al., Science 282:1075-1079, 1998; Ray et al., Mol Med Today 4:151-157, 1998, both of which are incorporated herein by reference), and the activity of all of them results in increased Aβ deposition, a very compelling argument in favor of the amyloid hypothesis.
The first of the 3 FAD genes codes for the Aβ precursor, APP (Selkoe, J Biol Chem 271:18295-18298, 1996, which is incorporated herein by reference). Mutations in the APP gene are very rare, but all of them cause AD with 100% penetrance and result in elevated production of either total Aβ or Aβ42, both in vitro (transfected cells) and in vivo (transgenic animals). The other two FAD genes code for presenilin 1 and 2 (PS1, PS2) (Hardy, Trends Neurosci 20:154-159, 1997, which is incorporated herein by reference). The presenilins contain 8 transmembrane domains and several lines of evidence suggest that they are involved in intracellular protein trafficking, although their exact function is still unknown. Mutations in the presenilin genes are more common than in the APP genes, and all of them also cause FAD with 100% penetrance. In addition, in vitro and in vivo studies have demonstrated that PS1 and PS2 mutations shift APP metabolism, resulting in elevated Aβ42 production. For a recent review on the genetics of AD, see Lippa, J Mol Med 4:529-536, 1999, which is incorporated herein by reference.
In spite of these compelling genetic data, it is still unclear whether Aβ generation and amyloid deposition are the primary cause of neuronal death and synaptic loss observed in AD. Moreover, the biochemical events leading to Aβ production, the relationship between APP and the presenilins, and between amyloid and neurofibrillary tangles are poorly understood. Thus, the picture of interactions between the major Alzheimer proteins is very incomplete, and it is clear that a large number of novel proteins are yet to be discovered.
The diagnosis of Alzheimer's disease at autopsy is definitive. Gross pathological changes are found in the brain, including low weight and generalized atrophy of both the gray and white matter of the cerebral cortex, particularly in the temporal and frontal lobes (Adams & Victor, 1977, Principles of Neurology, pp. 401-407 and Merritt, 1979, A Textbook of Neurology, 6th edition, Lea & Febiger, Philadelphia, pp. 484-489, both of which are incorporated herein by reference). The histological changes include neurofibrillary tangle (Kidd, Nature 197:192-193, 1963; Kidd, Brain 87:307-320, 1964, both of which are incorporated herein by reference), which consists of a tangled mass of paired helical and straight filaments in the cytoplasm of affected neurons (Oyanagei, Adv. Neurol. Sci. 18:77-88, 1979 and Grundke-Iqbal et al., Acta Neuropathol. 66:52-61, 1985, both of which are incorporated herein by reference).
The diagnosis of Alzheimer's disease during life is more difficult than at autopsy since the diagnosis depends upon inexact clinical observations. In the early and middle stages of the disease, the diagnosis is based on clinical judgment of the attending physician. In the late stages, where the symptoms are more recognizable, clinical diagnosis is more straightforward. But, in any case, before an unequivocal diagnosis can be made, other diseases, with partially overlapping symptoms, must be ruled out. Usually a patient must be evaluated on a number of occasions to document the deterioration in intellectual ability and other signs and symptoms. The necessity for repeated evaluation is costly, generates anxiety, and can be frustrating to patients and their families. Furthermore, the development of an appropriate therapeutic strategy is hampered by the difficulties of rapid diagnosis, particularly in the early stages where early intervention could leave the patient with significant intellectual capacity and a reasonable quality of life. In brief, no unequivocal laboratory test specific for Alzheimer's disease has been reported.
Alzheimer's disease is associated with degeneration of cholinergic neurons, in the basal forebrain, which play a fundamental role in cognitive functions, including memory (Becker et al., Drug Development Research 12:163-195, 1988, which is incorporated herein by reference). Progressive, inexorable decline in cholinergic function and cholinergic markers in the brain of Alzheimer's disease patients has been observed in numerous studies, and includes, for example, a marked reduction in acetylcholine synthesis, choline acetyltransferase activity, acetylcholinesterase activity, and choline uptake (Davis, Brain Res. 171:319-327, 1979 and Hardy et al., Neurochem. Int. 7:545-563, 1985, which are incorporated herein by reference). Even more, decreased cholinergic function may be an underlying cause of cognitive decline seen in Alzheimer's-disease patients (Kish et al., J. Neurol., Neurosurg., and Psych 51:544-548, 1988, which is incorporated herein by reference). Choline acetyltransferase and acetylcholinesterase activities decrease significantly as plaque count rises, and, in demented subjects, the reduction in choline acetyl transferase activity was found to correlate with intellectual impairment (Perry, et al., Brit. Med. J. 25, Nov. 1978, p. 1457, which is incorporated herein by reference).
Nerve cells produce nerve growth factors, proteins that regulate cell maturation during prenatal development and also play an important role in cell survival, repair, and regeneration during adult life. Because of their significance in cell maintenance and repair, these factors have attracted attention as potential treatments in Alzheimer's disease, stroke, spinal cord injury, and other neurodegenerative conditions. However, nerve growth factors are usually too large to cross the blood-brain barrier (BBB), a protective shield that restricts passage of molecules to the brain.
The BBB is functionally situated at the brain capillaries endothelium layer and covers a surface area of 12 m2/g of brain parenchyma. The total length of this capillary network is 650 km. The cerebral capillary endothelial cell displays some peculiar morphologic characteristics that form the anatomic basis of the blood-brain barrier. It differs from the peripheral capillary endothelial cell (referring to all non-CNS sites) in a number of ways:                First, the CNS endothelial cell layer is not fenestrated. Cells are joined by tight junctions composed of 6 to 8 pentalaminar structures. They actively block protein movements, hydrophilic transfer and even ionic diffusion. Thus, there is very little movement of compounds between endothelial cells from the blood to the CNS.        Second, and in contrast to the peripheral capillary endothelial cell, transcellular movement of molecules through the non-specific mechanism of fluid-phase endocytosis is generally absent. The cerebral vascular endothelial cell possesses a transcellular lipophilic pathway, allowing diffusion of small lipophilic compounds. In addition to this route, specific receptor-mediated transport systems are present for given molecules, like insulin, transferrin, glucose, purines and amino acids. These transport systems are highly selective and asymmetric.        Third, the CNS endothelial cell displays a net negative charge at its endoluminal side and at the basement membrane. This provides an additional selective mechanism by impeding anionic molecules to cross the membrane.        Fourth, the cerebral endothelial cell has very few pinocytic vesicles, and these vesicles are not involved in any transport function.        Fifth, astrocyte foot processes surround the microvascular endothelium and cover more than 95 percent of its surface, therefore interposing between capillaries and cerebral neuropil.        
By virtue of this selective barrier, the CNS can preferentially regulate the extracellular concentration of certain solutes, growth factors and neurotransmitters, keep certain molecules in the CNS and isolate itself from some others, and further isolate itself from sudden systemic homeostatic changes. It is therefore an integral component of the mechanisms involved in the tight regulation of the extra-cellular homeostasis necessary to the normal CNS function. This relatively impermeable barrier has some drawbacks, however, when considering the therapeutic delivery of a molecule to the CNS.
The delivery of therapeutic molecules across the BBB has proven to be a major obstacle in treating various brain disorders. The normal blood-brain barrier prevents passage of ionized water-soluble compounds with a molecular weight greater than 180 Daltons. Therefore, the BBB is a major impediment to the treatment of CNS diseases as many drugs are unable to reach this organ at therapeutic concentrations. More than 98% of the CNS-targeted drugs do not cross the BBB. Example of such disorders are: primary brain tumors, metastatic brain tumors, AD, addiction, ALS, head injury, Huntington's disease, multiple sclerosis (MS), depression, Cerebral Palsy, schizophrenia, epilepsy, stress and anxiety. Many new neurotherapeutic agents are being discovered, but because of a lack of suitable strategies for drug delivery across the BBB, these agents are ineffective. Such drugs will only become effective if strategies for brain delivery are developed in parallel.
Apart from molecular parameters, the permeability of the BBB and active transport mechanisms, a major determinant of molecular transport across the BBB is their concentration gradient—between the CNS and the cerebral circulation.
Additionally, the functioning BBB inhibits clearance of neurotoxic compounds, such as β-Amyloid, tau, PS1, and PS2, from the CNS into the systemic circulation. These neurotoxic compounds are therefore not metabolized and removed from the body to the extent desired, and therefore continue to have undesired effects in the CNS.
U.S. Pat. No. 5,752,515 to Jolesz et al., which is incorporated herein by reference, describes apparatus for image-guided ultrasound delivery of compounds through the blood-brain barrier. Ultrasound is applied to a site in the brain to effect in the tissues and/or fluids at that location a change detectable by imaging. At least a portion of the brain in the vicinity of the selected location is imaged, e.g., via magnetic resonance imaging, to confirm the location of that change. A compound, e.g., a neuropharmaceutical, in the patients bloodstream is delivered to the confirmed location by applying ultrasound to effect opening of the blood-brain barrier at that location and, thereby, to induce uptake of the compound there.
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